The present invention relates to methods and compositions for reducing immune rejection, for example, transplant- or autoimmune disorder-related immune rejection. The present invention also relates to methods and compositions for monitoring transplant acceptance and for monitoring an autoimmune disorder in a subject mammal. The present invention still further relates to methods for identifying compounds that can reduce immune rejection.
The present invention is based, in part, on the discovery, demonstrated herein, that immune rejection can be monitored by determining the amount of particular members of the Jak/Stat signal transduction pathway present within an affected tissue (that is, a transplant cell, tissue, organ, or organ system, or a cell, tissue, organ, or organ system that is, or is suspected of, being affected by an autoimmune disorder). The present invention is further based, in part, on the discovery, demonstrated herein, that immune rejection can be reduced and tolerance can be induced by modulating the amount of these particular members of the Jak/Stat signal transduction pathway present, expressed or active within an affected tissue. In particular, the results presented herein demonstrate that immune rejection can be monitored by determining the amount of Stat1 mRNA or protein, Stat2 mRNA or protein, Stat3 mRNA, protein Stat4 mRNA or protein, Stat6 mRNA or protein, SOCS1 mRNA or protein, or SOCS3 mRNA or protein present, e.g., present in an affected tissue.
Ongoing advances in transplantation, including new immunosuppressive agents and improvements in histocompatibility matching, organ procurement, and surgical techniques, are gradually improving the outcome of clinical transplantation (Hariharan et al, 2000. N Engl J Med 342:605-12). However, chronic allograft rejection remains the prime determinant of long-term graft survival (Paul. L. C., 1999, Kidney International 56:783-793).
Tissue transplantation between genetically nonidentical individuals results in immunological rejection of the tissue through T cell-dependent mechanisms. To prevent allograft rejection, immunosuppressive agents such as calcineurin phosphatase inhibitors and glucocorticosteroids which directly or indirectly interfere with IL-2 signaling are administered to transplant recipients (see, e.g., Borel, J. F., 1989, Pharmacol. Rev. 42:260-372; Morris, P. J., 1991, Curr. Opin. Immunol. 3:748-751; Sigal et al., 1992, Ann. Rev. Immunol. 10:519-560; and L""Azou et al., 1999, Arch. Toxicol. 73:337-345). The most commonly used immunosuppressive agents today are cyclosporin A, FK506, and rapamycin. These immunosuppressive agents act indiscriminately on all T cells by impairing T cell receptor (xe2x80x9cTCRxe2x80x9d) signal transduction. Further, since the effect of the immunosuppressive agents is short-lasting, transplant recipients normally require life-long treatment of immunosuppressive agents to prevent transplant rejection. As a result of the long-term nonspecific immunosuppression, these immunosuppressive agents have many serious adverse effects. For example, the administration of cyclosporin A or FK506 to a transplant recipient results in degenerative changes in renal tubules. Transplant recipients receiving long-term immunosuppressive treatment have a high risk of developing infections and tumors. For example, patients receiving immunotherapy are at higher risk of developing lymphomas, skin tumors and brain tumors (see, e.g., Fellstrom et al., 1993, Immunol. Rev. 134:83-98).
An alternative to immunosuppressive agents for the prevention of allograft rejection is the blockage of specific receptors involved in T cell costimulation. T cell activation requires both TCR-mediated signal transduction and simultaneously delivered costimulatory signals. These costimulatory signals are contributed, in part, by the activation of the costimulatory molecule CD28, which is expressed on resting T cells, by CD80 (B7-1) or CD86 (B7-2) expressed on antigen presenting cells (APCs). The activation of the costimulatory molecule CD40, which is expression on antigen presenting cells (i.e., B cells, dendritic cells, and macrophages), by CD40 ligand (xe2x80x9cCD40Lxe2x80x9d), which is expressed on activated T cells, contributes to the upregulation of T cell activation by inducing the expression of B7-1 and B7-2 on antigen presenting cells and the production of certain chemokines and cytokines such as IL-8, MIP-1xcex1, TNF-xcex1, and IL-12 (Cella et al., 1996, J. Exp. Med. 184:747-752: and Caux et al., 1994, J. Exp. Med. 180:1263-1272). The CD40/CD40L interaction also results in the differentiation of T cells to T helper (xe2x80x9cTHxe2x80x9d) type 1 cells in part due to the expression of cytokines such as IL-12 by dendritic cells and macrophages.
CTLA-4 is normally expressed as a membrane-bound receptor on T cells and has been shown to downregulate T cell activation by competing with CD28 for B7-1 and B7-2. The administration of soluble CTLA-4Ig is believed to prevent allograft rejection by competing with CD28 for B7-1 and B7-2. Soluble CTLA-4Ig has been administered to transplant recipients to disrupt the CD28/B7 interaction so that T cell costimulation is blocked and allograft rejection does not occur (Zheng et al., 1999, J. Immunol. 162:4983-4990; Lenschow et al., 1996, Ann. Rev. Immunol. 14:233-258). Unfortunately, CTLA-4Ig has variable efficacy, and typically does not prevent development of chronic rejection.
Anti-CD40L (anti-CD154) monoclonal antibodies have also been administered to transplant recipients to prevent allogaft rejection. These antibodies function by blocking the interaction of CD40 on antigen presenting cells (APC) and CD40L on activated T cells. It has recently been shown that graft survival achieved through the use of anti-CD40L monoclonal antibodies results in a significant inhibition of TH1 type cytokines (i.e., IL-2, IL-12, TNFxcex1, and IFNxcex3), and an increase in the levels of the TH2 type cytokines (i.e., IL-4, and IL-10) in the graft sections (Hancock et al., 1996, Proc. Natl. Acad. Sci. USA 93:13967-13972). Although the administration of anti-CD40L monoclonal antibodies has been shown to result in permanent graft survival when given to mice in combination with donor-specific spleen cells, adverse side effects such as coagulation have also been shown to be associated with the administration of anti-CD40L monoclonal antibodies. Initial clinical trials in adult renal transplant recipients receiving anti-CD40L monoclonal antibody plus glucocorticoids were halted because of thromboembolic complications (Vincent, J., Biogen News, press release, Nov. 2, 1999, www.prnewswire.com), though the extent to which thromoboembolism was attributable to monoclonal antibodies versus non-specific factors in the antibody formulation is unclear (Kawai et al., 2000, Nature Med. 6:114; and Kirk et al., 2000, Nature Med. 6:114). Further, in the primate renal allograft study, concomitant use of mainstream immunosuppressive agents such as FK-506, methylprednisolone and mycophenolate mofetil diminished the efficacy of CD40L (CD154) mAb, though the exact contribution of each of the individual drugs to this reduction in efficacy was not determined (Kirk, A. D., 1999, Nature Medicine 5:686-693.). The results presented herein demonstrate that some, but not all, combinations of CD154 mAb and immunosuppressive agents are antagonistic, and that strategies for design of clinical trials based on use of CD154 mAb can be logically developed by taking into account the extent to which a given drug inhibits induction of CD154.
In addition, no satisfactory methods presently exist for monitoring whether a transplant graft is being accepted or rejected by a recipient. In general, signs of cellular damage within the transplant tissue can be assayed. Alternatively, for tissues such as kidney or liver, physiological function of the transplant tissue can be assayed. Often, however, by the time overt signs of either cellular damage or a decrease in physiological function are detected, the tissue graft is already beyond rescue. This is particularly true in the case of such organ transplants as heart transplants, with which the first overt signs of rejection are often complete failure of the heart""s function.
Accordingly, there is a need for improved, safer immunomodulatory treatments that have long-lasting effects for the prevention of transplant rejection. In particular, there is a need for treatments that are more specific and less toxic than the currently available therapeutic agents. Further, there is also a great need for an improved method for monitoring acceptance of transplant tissue in subject mammals that have undergone a transplant.
Signal transduction pathways represent molecular solutions to the fact that such molecules as polypeptide hormones, growth factors and cytokines cannot cross the cell membrane, but must activate intracellular signaling molecules to elicit a response in target cells. Among such signal transduction pathways is the Jak/Stat signal transduction pathway. See, e.g., Heim, M. H., 1999, J. Recept. and Sig. Trans. Res. 19:75-120; and Leonard, W. J. and O""Shea, J. J., 1998, Ann. Rev. Immunol. 16:293-322.
While the pathway was originally discovered as part of a study of interferon-induced intracellular signalling, to date, several dozen polypeptide ligands have been identified that activate the Jak/Stat pathway. Defects in the Jak/Stat pathway have been identified in a number of diseases, including leukemias, lymphomas, inherited immunodeficiency syndromes, breast cancer and a form of dwarfism caused by constitutively activation of a Stat by a mutant fibroblast growth factor-receptor.
Stats (Signal transducers and activators of transcription) are phosphoproteins that are transcription factors, and that are activated in response to cytokines, growth factors and interferons. Stats are activated by receptor-associated Janus kinases (xe2x80x9cJaksxe2x80x9d), which include Jak1, Jak2, Tyk2, and Jak3. Specifically, a ligand-induced receptor aggregation results in the transphorphorylation and activation of the catalytic activity of the associated Jak. The activated Jak phosphorylates the receptors at multiple sites. Stats are recruited to the multimeric complex consisting of the phosphorylated receptor and catalytically active Jak. The catalytically active Jak phosphorylates tyrosine residues in the carboxy-terminus of the Stats. The phosphorylated Stats form homodimers and heterodimers (Darnell, J. E., 1997, Science 277:1630-1635; and Leonard et al., 1998, Ann. Rev. Immunol. 16:293-322; and Darnell et al., 1994, Science 264:1415-1421). The dimerization of Stats is believed to trigger the dissociation of Stats from the receptor complex and their translocation to the nucleus. In the nucleus, Stat dimers bind to their cognate DNA regulatory elements, which binding results in increased transcription, i.e., transactivation. Thus, the Jak/Stat system provides a method of both signal amplification and transduction.
Seven Stat genes (Stat1, Stat2, Stat3, Stat4, Stat5A, Stat5B, and Stat6) and several Stat isoforms have been discovered, the isoforms resulting from alternative splicing or posttranslational processing (for review see, e.g., Leonard et al., 1998, Ann. Rev. Immunol. 16:293-322). Different Stats are activated in response to different cytokines and growth factors. For example, Stat4 has been shown be activated in response to IL-12 induced signal transduction (Thierfelder et al., 1996, Nature 382:171-174; and Kaplan et al., 1996, Nature 382:174-177). Stat6 has been shown to be activated in response to IL-4 and IL-13 induced signal transduction (Takeda et al., 1996, Nature 380:627-630). Certain transcription factors activated in response to a given cytokine have been shown to be important in TH1 and/or TH2 differentiation. Stat4 has been shown to be important in TH1 differentiation and Stat6 has been shown to be important in TH2 differentiation (see, e.g., Romagnani, S., 1997, Immunology Today 18:263-266; Ray, A. and Cohn, L., 1999, J. Clin. Invest. 104(8):985-993).
With respect to TH1 and TH2, the majority of mature T lymphocytes can be divided into two distinct phenotypes: CD8+ cytotoxic T lymphocytes (CTLs), which display the CD8 marker on their cell surface, and CD4+ helper T lymphocytes (T helper or TH cells), which display the CD4 marker on their cell surface. TH cells are involved in both humoral (i.e., antibody) and cell-mediated forms of immune response. TH cells have been further categorized into two distinct subpopulations, termed TH1 and TH2 cell subpopulations. These two subpopulations of TH cells have been categorized on the basis of their restricted cytokine profiles and different functions. For example, TH1 cells are known to produce IL-2, IL-12, tumor necrosis factor xcex2 (xe2x80x9cTNF-xcex2xe2x80x9d), and interferon-xcex1 (xe2x80x9cIFN-xcex1xe2x80x9d). TH2 cells are known to produce IL-4, IL-5, IL-10 and IL-13. Inappropriate immune responses have been shown to be associated with various diseases and disorders. For example, an inappropriate TH2-like response has shown to be associated with atopic conditions, such as asthma and allergy (see, e.g. , Holgate, S. T., 1997, Lancet 350(suppl. II):5-9; Ray, A. and Cohn, L, supra; Oettgen, H. C. and Geha, R. S., 1999, J. Clin. Invest. 104(7):829-835). Further, an inappropriate TH 1-like response has been shown to associated with the pathogenesis of autoimmune diseases such multiple sclerosis, pancreases of insulin-dependent diabetes patients, thyroid glands of Hashimoto""s thyroiditis, and gut of Crohn""s disease patients.
Three protein families have been discovered that negatively regulate cytokine-induced Jak/Stat signaling, tyrosine phosphatases SHP1 and SHP2, the suppressors of cytokine signaling (xe2x80x9cSOCSxe2x80x9d), and protein inhibitors of activated Stats (PIAS). SHP1 and SHP2 bind to phosphorylated tyrosine residues on receptors or Jaks, and inactivate signaling by dephosphorylating them (Haque et al., 1998, J. Biol. Chem. 273:33898-33896; and You et al., 1999, Mol. Cell. Biol. 19:2416-2424).
The SOCS family of proteins have been shown to inhibit the Jak/Stat pathway by inhibiting the activity of the Jaks (Hilton et al., Proc. Natl. Acad. Sci. USA 95:114-119; and Hilton, 1999, Cell. and Mol. Life Sci. 55:1658-1577). The nature of the interaction between the different receptors, Jaks, and the SOCS is unclear (Hilton, D. J., 1999, Cell. Mol. Sci. 55:1568-1577). SOCS1 have been shown to directly interact with all the Jaks and Tyk2. CIS (Cytokine inducible SH2 containing protein), a member of the SOCS family, on the other hand, was shown to interact with the EPO receptor or the xcex2 chain of the IL-3 receptor in a phosphorylation dependent manner, indicating it may act by competing with Stat molecules for binding to receptors (Yoshimura et al., 1995, EMBO J. 14:2816-2826). SOCS1 expression inhibits IL-6, LIF, oncostatin M, IFN-xcex3, IFN-xcex2, IFN-xcex1, thrombopoeitin, and growth hormone (GH) induced Jak/Stat signaling. SOCS3 expression inhibits IFN-xcex3, IFN-xcex2, IFN-xcex1, GH and leptin.
Four members of the PIAS family have been identified, PIAS1, PIAS3, PIASxxcex1, and PIASxxcex2. PIAS1 was found to bind only to activated Stat1, and PIAS3 to only activated Stat3 (Liu et al., 1998, Proc. Natl. Acad. Sci. USA 95:10626-10631; and Chung et al., 1997, Science 278:1803-1805). PIAS-mediated inhibition of the Jak/Stat signaling pathway, unlike SOCS-mediated inhibition of the Jak/Stat signaling pathway, is very specific. However, unlike some of the SOCS which are elevated rapidly in response to cytokines, the PIAS levels in the cells are more or less constant.
The present invention relates to methods and compositions for reducing immune rejection, for example, transplant- or autoimmune disorder-related immune injury or rejection. The present invention also relates to methods and compositions for monitoring transplant acceptance and for monitoring an autoimmune disorder in a subject mammal. The present invention still further relates to methods for identifying compounds that can reduce immune injury.
The present invention is based, in part, on the discovery, demonstrated herein, that immune rejection can be monitored by determining the amount of particular members of the Jak/Stat signal transduction pathway present within an affected tissue (that is, a transplant cell, tissue, organ, or organ system, or a cell, tissue, organ, or organ system that is, or is suspected of, being affected by an autoimmune disorder). In particular, the results presented herein demonstrate that immune rejection can be monitored by determining the amount of Stat4 mRNA or protein, Stat6 mRNA or protein, SOCS1 mRNA or protein, or SOCS3 mRNA or protein, present in an affected tissue. The results presented herein also demonstrate that immune rejection can be monitored by determining the amount of Stat1 mRNA or protein, Stat2 mRNA or protein, or Stat3 mRNA or protein present, e.g., present in an affected tissue. The present invention is further based, in part, on the discovery, demonstrated herein, that immune rejection can be reduced and tolerance can be induced by modulating the amount of these particular members of the Jak/Stat signal transduction pathway present, expressed or active within an affected tissue.
Thus, in one aspect, the invention relates to methods for monitoring acceptance of a transplant in a subject mammal that has undergone a transplant, wherein said method comprises: determining the amount of at least one of the following: (i) Stat4 mRNA or Stat4 protein, (ii) Stat6 mRNA or Stat6 protein, (iii) SOCS1 mRNA or SOCS1 protein, or (iv) SOCS3 mRNA or SOCS3 protein, present in a transplant sample from the subject. In alternate embodiments, such methods comprise determining the amount of at least two, at least three, or each of (i) to (iv) present in the transplant sample. In certain embodiments, the amount of mRNA is determined, and can, for example, be determined via use of nucleic acid microarrays. In other embodiments, the amount of protein is determined, while in still other embodiments, the amount of mRNA and protein is determined. With respect to Stat6, when the amount of Stat6 is being determined, it is preferable that the amount of Stat6 protein be determined. In any such embodiment wherein a Stat protein amount is determined, the amount determined can be the total amount of the Stat protein present in a sample or, alternatively, can be the amount of phosphorylated Stat protein present in the sample.
In a preferred embodiment, a method for monitoring acceptance of a transplant in a subject mammal that has undergone a transplant comprises determining the amount of Stat4 and Stat6 mRNA or Stat4 and Stat6 protein present in a transplant sample from the subject. Such an embodiment can further comprise determining the ratio of Stat4 to Stat6 amounts.
The methods for monitoring acceptance of a transplant in a subject mammal that has undergone a transplant can further comprise assaying the transplant sample for evidence of lymphocyte infiltration or tissue damage (cell injury) using standard techniques. For example, histological techniques well known to those of skill in the art can be utilized to evaluate internationally recognized and used diagnostic criteria for the evaluation of graft rejection, which include features specific for each organ involved. For example, immunohistologic evaluation of such tissues, via, e.g., use of labeled antibody techniques to localize and quantitate gene expression. The evaluation of such criteria can, therefore, be enhanced by, for example, localization of Stat4, Stat6, SOCS1 and/or SOCS3 proteins, and/or detection of corresponding mRNAs via, e.g., in situ hybridization.
Such methods can also further comprise comparing the amount or ratio determined to that present in a control sample, for example, a corresponding pre-transplant subject sample or a subject blood sample. In instances wherein the amount of Stat4, SOCS1, or SOCS3 mRNA or protein in the transplant sample is greater than, or the amount of Stat6 mRNA or protein in the transplant sample is less than, that of the control sample, such a result indicates that acceptance of the transplant has not occurred, has not been induced or is not being maintained. In instances wherein the amount of Stat4, SOCS1, or SOCS3 mRNA or protein in the transplant sample is less than, or the amount of Stat6 mRNA or protein in the transplant sample is equal to or greater than that of the control sample, such a result indicates that acceptance of the transplant has occurred, is being induced or is being maintained. In instances wherein the ratio of Stat4 to Stat6 in the transplant sample is greater than or equal to that in the control sample, such a result indicates that acceptance of the transplant has not occurred, has not been induced or is not being maintained. In instances wherein the ratio of Stat4 to Stat6 in the transplant sample is less than that in the control sample, such a result indicates that acceptance of the transplant has occurred, has been induced or is being maintained.
In another aspect, the invention relates to methods for monitoring acceptance of a transplant in a subject mammal that has undergone a transplant, wherein said method comprises: determining the amount of at least one of the following: (i) Stat1 mRNA or Stat1 protein, (ii) Stat2 mRNA or Stat2 protein, or (iii) Stat3 mRNA or Stat3 protein, present in a cell sample from the subject. In alternate embodiments, such methods comprise determining the amount of at least two or each of (i) to (iii) present in the sample. In certain embodiments, the amount of mRNA is determined, and can, for example, be determined via use of nucleic acid microarrays. In other embodiments, the amount of protein is determined, while in still other embodiments, the amount of mRNA and protein is determined. In any such embodiment wherein a Stat protein amount is determined, the amount determined can be the total amount of the Stat protein present in a sample or, alternatively, can be the amount of phosphorylated Stat protein present in the sample.
In a particular embodiment of such Stat 1-, Stat 2-, and/or Stat 3-related methods, the cell sample is a transplant sample obtained within 2 to 3 days post-transplantation. In an alternative embodiment of such Stat 1-, Stat 2-, and/or Stat 3-related methods, the cell sample is a subject blood sample.
Such Stat 1-, Stat-2, and/or Stat 3-related methods can also further comprise comparing the amount determined to that present in a control sample, for example, a corresponding pre-transplant subject sample or, in the case of embodiments wherein the cell sample is a transplant sample obtained within 2-3 days post-transplantation, a subject blood sample. In instances wherein the amount of Stat1, Stat2, or Stat3 mRNA or protein in the cell sample is greater than that of the control sample, such a result indicates that acceptance of the transplant has not occurred, has not been induced or is not being maintained. In instances wherein the amount of Stat1, Stat2, or Stat3 mRNA or protein in the transplant sample is less than that of the control sample, such a result indicates that acceptance of the transplant has occurred, is being induced or is being maintained.
In another aspect, the invention relates to methods for monitoring an autoimmune disorder in a subject mammal, wherein said method comprises: determining the amount of at least one of the following: (i) Stat4 mRNA or Stat4 protein, (ii) Stat6 mRNA or Stat6 protein, (iii) SOCS1 mRNA or SOCS1 protein, or (iv) SOCS3 mRNA or SOCS3 protein, present in a sample from a subject mammal being treated for or suspected of exhibiting the autoimmune disorder, wherein the sample is obtained from a tissue affected by the disorder. In alternate embodiments, such methods comprise determining the amount of at least two, at least three, or each of (i) to (iv) present in the sample. In certain embodiments, the amount of mRNA is determined, and can, for example, be determined via use of nucleic acid microarrays. In other embodiments, the amount of protein is determined, while in still other embodiments, the amount of mRNA and protein is determined. With respect to Stat6, when the amount of Stat6 is being determined, it is preferable that the amount of Stat6 protein be determined. In any such embodiment wherein a Stat protein amount is determined, the amount determined can be the total amount of the Stat protein present in a sample or, alternatively, can be the amount of phosphorylated Stat protein present in the sample.
In a preferred embodiment, a method for monitoring an autoimmune disorder in a subject mammal comprises determining the amount of Stat4 and Stat6 mRNA or Stat4 and Stat6 protein present in a sample from the subject mammal being treated for or suspected of exhibiting the autoimmune disorder, wherein the sample is obtained from a tissue affected by the disorder. Such an embodiment can further comprise determining the ratio of Stat4 to Stat6 amounts.
The methods for monitoring an autoimmune disorder in a subject mammal can further comprise assaying the sample for evidence of leukocyte infiltration or tissue damage (cell injury) using standard techniques. For example, histological techniques well known to those of skill in the art can be utilized. Alternatively, standard techniques can be utilized to assay (e.g., in serum) for the presence of autoimmune antibodies associated with the particular autoimmune disorder of interest. There are internationally used diagnostic criteria for evaluation of graft rejection, with features specific for each organ. The immunohistologic evaluation of such tissues, i.e., use of unlabeled-antibody techniques to localize and quantitate gene expression, can be enhanced by localization of Stat4 and Stat6 proteins, or detection of corresponding mRNAs by in situ hybridization.
Such methods for monitoring an autoimmune disorder in a subject mammal can further comprise comparing the amount or ratio determined to that present in a control sample, for example, a corresponding tissue not affected by the disorder or a subject blood sample. In instances wherein the amount of Stat4, SOCS1, or SOCS3 mRNA or protein in the sample is greater than, or the amount of Stat6 mRNA or protein in the sample is less than, that of the control sample, such a result indicates that the subject mammal exhibits or continues to exhibit the disorder. In instances wherein the amount of Stat4, SOCS1, or SOCS3 mRNA or protein in the sample is less than, or the amount of Stat6 mRNA or protein in the sample is equal to or greater than that of the control sample, such a result indicates that the subject mammal does not exhibit the disorder or that treatment for the disorder is effective. In instances wherein the ratio of Stat4 to Stat6 in the sample is greater than or equal to that in the control sample, such a result indicates that the subject mammal exhibits or continues to exhibit the disorder. In instances wherein the ratio of Stat4 to Stat6 in the transplant sample is less than that in the sample, such a result indicates that the subject mammal does not exhibit the disorder or that treatment for the disorder is effective.
The methods for monitoring transplant acceptance or monitoring an autoimmune disorder can be performed with kits designed for carrying out such methods. As such, the present invention also relates to kits for monitoring transplant acceptance and autoimmune disorders.
In yet another aspect, the present invention relates to a method for identifying a compound to be tested for an ability to reduce immune rejection, said method comprises: (a) contacting an activated T cell sample with a test compound; (b) determining the amount of at least one of the following: (i) Stat1 mRNA or Stat1 protein, (ii) Stat2 mRNA or Stat2 protein, (iii) Stat3 mRNA or Stat3 protein, (iv) Stat4 mRNA or Stat4 protein, (v) Stat6 mRNA or Stat6 protein; (vi) SOCS1 mRNA or SOCS1 protein, or (vii) SOCS3 mRNA or SOCS3 protein, present in (a); and (c) comparing the amount(s) in (a) to that/those present in a corresponding control activated T cell sample that has not been contacted with the test compound, so that if the amount of (i), (ii), (iii), (iv), (vi), or (vii) is decreased, or the amount of (v) is increased, relative to the amount in the control sample, a compound to be tested for an ability to reduce immune rejection is identified. In alternate embodiments, such methods comprise determining the amount of at least two, at least three, at least four, at least five, at least six, or each of (i) to (vii) present in the activated T cell sample and comparing the amounts to those present in the control sample.
In certain embodiments, the amount of mRNA is determined, in other embodiments, the amount of protein is determined, while in still other embodiments, the amount of mRNA and protein is determined. With respect to Stat6, when the amount of Stat6 is being determined, it is preferable that the amount of Stat6 protein be determined. In any such embodiment wherein a Stat protein amount is determined, the amount determined can be the total amount of the Stat protein present in a sample or, alternatively, can be the amount of phosphorylated Stat protein present in the sample.
In a preferred embodiment of a method for identifying a compound to be tested for an ability to reduce immune rejection, said method comprises: (a) contacting an activated T cell sample with a test compound; (b) determining the amount of Stat4 mRNA and Stat6 mRNA or Stat4 protein and Stat6 protein present in the sample; and (c) comparing the amounts in (b) to those present in a corresponding control activated T cell sample that has not been contacted with the test compound, so that if the amount of Stat4 is decreased or the amount of Stat6 is increased relative to the amount in the control sample, a compound to be tested for an ability to reduce immune rejection is identified.
In another preferred embodiment of a method for identifying a compound to be tested for an ability to reduce immune rejection, said method comprises: (a) contacting an activated T cell sample with a test compound; (b) determining the ratio of Stat4 mRNA to Stat6 mRNA or Stat4 protein to Stat6 protein present in the sample; and (c) comparing the ratio in (b) to that present in a corresponding control activated T cell sample that has not been contacted with the test compound, so that if the ratio in the sample is decreased relative to that in the control sample, a compound to be tested for an ability to reduce immune rejection is identified.
In another aspect, the present invention relates to a method for identifying a compound to be tested for an ability to reduce immune rejection, said method comprises: (a) contacting a resting T cell sample, a T cell activator and a test compound; (b) determining the amount of at least one of the following: (i) Stat1 mRNA or Stat1 protein, (ii) Stat2 mRNA or Stat2 protein, (iii) Stat3 mRNA or Stat3 protein, (iv) Stat4 mRNA or Stat4 protein, (v) Stat6 mRNA or Stat6 protein; (vi) SOCS1 mRNA or SOCS1 protein, or (vii) SOCS3 mRNA or SOCS3 protein, present in (a); and (c) comparing the amount(s) in (a) to that/those present in a corresponding resting T cell sample that has been contacted with the T cell activator, but has not been contacted with the test compound, so that if the amount of (i), (ii), (iii), (iv), (vi), or (vii) is decreased, or the amount of (v) is increased, relative to the amount in the control sample, a compound to be tested for an ability to reduce immune rejection is identified. In alternate embodiments, such methods comprise determining the amount of at least two, at least three, at least four, at least five, at least six, or each of (i) to (vii) present in the activated T cell sample and comparing the amounts to those present in the control sample.
In certain embodiments of such methods, the amount of mRNA is determined, in other embodiments, the amount of protein is determined, while in still other embodiments, the amount of mRNA and protein is determined. With respect to Stat6, when the amount of Stat6 is being determined, it is preferable that the amount of Stat6 protein be determined. In any such embodiment wherein a Stat protein amount is determined, the amount determined can be the total amount of the Stat protein present in a sample or, alternatively, can be the amount of phosphorylated Stat protein present in the sample. Further, in certain embodiments, the resting T cell is a primary T cell, and in other embodiments, the resting T cell is a T cell line.
In a preferred embodiment of a method for identifying a compound to be tested for an ability to reduce immune rejection, said method comprises: (a) contacting a resting T cell sample, a T cell activator and a test compound; (b) determining the amount of Stat4 mRNA and Stat6 mRNA or Stat4 protein and Stat6 protein present in the sample; and (c) comparing the amounts in (b) to those present in a corresponding control resting T cell sample that has been contacted with the T cell activator, but has not been contacted with the test compound, so that if the amount of Stat4 is decreased or the amount of Stat6 is increased relative to the amount in the control sample, a compound to be tested for an ability to reduce immune rejection is identified.
In another preferred embodiment of a method for identifying a compound to be tested for an ability to reduce immune rejection, said method comprises: (a) contacting a resting T cell sample, a T cell activator and a test compound; (b) determining the ratio of Stat4 mRNA to Stat6 mRNA or Stat4 protein to Stat6 protein present in the sample; and (c) comparing the ratio in (b) to that present in a corresponding control resting T cell sample that has been contacted with a T cell activator, but has not been contacted with the test compound, so that if the ratio in (a) is decreased relative to that in the control sample, a compound to be tested for an ability to reduce immune rejection is identified.
In another aspect, the present invention relates to a method for identifying a compound to be tested for an ability to reduce immune rejection, comprising: (a) contacting a T cell sample, a cytokine and a test compound, wherein the T cell sample is responsive to the cytokine; (b) determining the amount of at least one of the following: (i) Stat1 mRNA or Stat1 protein, (ii) Stat2 mRNA or Stat2 protein, (iii) Stat3 mRNA or Stat3 protein, (iv) Stat4 mRNA or Stat4 protein, (v) Stat6 mRNA or Stat6 protein; (vi) SOCS1 mRNA or SOCS1 protein, or (vii) SOCS3 mRNA or SOCS3 protein, present in (a); and (c) comparing the amount(s) in (a) to that/those present in a corresponding control T cell sample that has been contacted with the cytokine, but has not been contacted with the test compound, so that if the amount of (i), (ii), (iii), (iv), (vi), or (vii) is decreased, or the amount of (v) is increased, relative to the amount in the control sample, a compound to be tested for an ability to reduce immune rejection is identified. In preferred embodiments, the cytokine is IL-2, IL-4, IL-12, or IL-13.
In certain embodiments of such methods, the amount of mRNA is determined, in other embodiments, the amount of protein is determined, while in still other embodiments, the amount of mRNA and protein is determined. With respect to Stat6, when the amount of Stat6 is being determined, it is preferable that the amount of Stat6 protein be determined. In any such embodiment wherein a Stat protein amount is determined, the amount determined can be the total amount of the Stat protein present in a sample or, alternatively, can be the amount of phosphorylated Stat protein present in the sample.
In a preferred embodiment of such a method for identifying a compound to be tested for an ability to reduce immune rejection, said method comprises: (a) contacting a T cell sample, a cytokine and a test compound, wherein the T cell sample is responsive to the cytokine; (b) determining the amount of Stat4 and Stat6 mRNA or Stat4 and Stat6 protein present in the sample; and (c) comparing the amounts in (a) to those present in a corresponding control T cell sample that has been contacted with the cytokine, but has not been contacted with the test compound, so that if the amount of Stat4 is decreased or the amount of Stat6 is increased relative to the amounts in the control sample, a compound to be tested for an ability to reduce immune rejection is identified.
In another preferred embodiment of such a method for identifying a compound to be tested for an ability to reduce immune rejection, said method comprises: (a) contacting a T cell sample, a cytokine and a test compound, wherein the T cell sample is responsive to the cytokine; (b) determining the ratio of Stat4 mRNA to Stat6 mRNA or Stat4 mRNA to Stat6 protein present in the sample; and (c) comparing the ratio to in (a) to that present in a corresponding control T cell sample that has been contacted with the cytokine, but has not been contacted with the test compound, so that if the ratio in the sample is decreased relative to that in the control sample, a compound to be tested for an ability to reduce immune rejection is identified.
In yet another aspect, the present invention relates to methods for reducing immune rejection in a subject mammal, said methods comprising: administering to a subject mammal in need of such a reduction a concentration of a compound sufficient to decrease the level of Stat4 mRNA or protein in the subject relative to that observed in the subject in the absence of the compound, wherein said compound does not induce platelet aggregation and does not affect NF-xcexaB activation in CD40L+ cells.
Alternatively, such methods for reducing immune rejection in a subject mammal can comprise: administering to a subject mammal in need of such a reduction a concentration of a compound sufficient to increase the level of Stat6 mRNA or protein in the subject relative to that observed in the subject in the absence of the compound, wherein said compound does not induce platelet aggregation and does not affect NF-xcexaB activation in CD40L+ cells.
Such methods for reducing immune rejection in a subject mammal can also comprise: administering to a subject mammal in need of such a reduction a concentration of a compound sufficient to decrease the level of Stat4 mRNA or protein and maintain or increase the level of Stat6 mRNA or protein in the subject relative to that observed in the subject in the absence of the compound, wherein said compound does not induce platelet aggregation and does not affect NF-xcexaB activation in CD40L+ cells.
The methods of the present invention for reducing immune rejection can be utilized, e.g., for reducing immune rejection in a subject mammal that has undergone a transplant. For example, such methods can induce tolerance in a subject mammal that has undergone a transplant. The methods of the present invention for reducing immune rejection can also be utilized, e.g., for reducing immune rejection in a subject mammal exhibiting an autoimmune disorder.
As used herein, the term xe2x80x9ctransplantxe2x80x9d includes any cell, organ, organ system or tissue which can elicit an immune response in a recipient subject mammal. In general, therefore, a transplant includes an allograft or a xenograft cell, organ, organ system or tissue. An allograft refers to a graft (cell, organ, organ system or tissue) obtained from a member of the same species as the recipient. A xenograft refers to a graft (cell, organ, organ system or tissue) obtained from a member of a different species as the recipient.
The term xe2x80x9cimmune rejection,xe2x80x9d as used herein, is intended to refer to immune responses involved in transplant rejection, as well as to the concomitant physiological result of such immune responses, such as for example, interstitial fibrosis, chronic graft artheriosclerosis, or vasculitis. The term xe2x80x9cimmune rejection,xe2x80x9d as used herein, is also intended to refer to immune responses involved in autoimmune disorders, and the concomitant physiological result of such immune responses, including T cell-dependent infiltration and direct tissue injury; T cell-dependent recruitment and activation of macrophages and other effector cells; and T cell-dependent B cell responses leading to autoantibody production.
The term xe2x80x9ctransplant rejection,xe2x80x9d as used herein, refers to T cell-mediated rejection of transplant cells, organs, organ systems or tissue. In general, such transplant rejection generally includes accelerated, acute and chronic rejection. It is intended that the term, as used herein, also refer to graft versus host disease, and the physiological results of such a disorder.
The term xe2x80x9creducing immune rejection,xe2x80x9d is meant to encompass prevention or inhibition of immune rejection, as well as delaying the onset or the progression of immune rejection. The term is also meant to encompass prolonging survival of a transplant in a subject mammal, or reversing failure of a transplant in a subject. Further, the term is meant to encompass ameliorating a symptom of an immune rejection, including, for example, ameliorating an immunological complication associated with immune rejection, such as for example, interstitial fibrosis, chronic graft atherosclerosis, or vasculitis. The term is also meant to encompass induction of tolerance in a subject mammal that has undergone a transplant.
The term xe2x80x9ctolerance,xe2x80x9d as used herein, refers to a state wherein the immune system of a transplant recipient subject mammal is non-responsive to the transplant. This state is considered donor transplant-specific, and, as such, is distinguished from nonspecific immunosuppression. Operatively, the term as used herein, refers to permanent acceptance of a graft without ongoing immunosuppression, wherein, for example, challenge with a second graft of donor origin (especially when the second graft is of the same tissue as the first graft) should be accepted, and challenge with a third party graft should be rejected.
The term xe2x80x9cautoimmune rejection,xe2x80x9d as used herein, refers to immune responses involved in autoimmune disorders, and the concomitant physiological result of such immune responses.
The term xe2x80x9cactivated T cell,xe2x80x9d as used herein, refers to a T cell that expresses antigens indicative of T-cell activation (that is, T cell activation markers). Examples of T cell activation markers include, but are not limited to, CD25, CD26, CD30, CD38, CD69, CD70, CD71, ICOS, OX-40 and 4-1BB. The expression of activation markers can be measured by techniques known to those of skill in the art, including, for example, western blot analysis, northern blot analysis, RT-PCR, immunofluorescence assays, and fluorescence activated cell sorter (FACS) analysis.
The term xe2x80x9cresting T cell,xe2x80x9d as used herein, refers to a T cell that does not express T-cell activation markers. Resting T cells include, but are not limited to, T cells which are CD2531 , CD69xe2x88x92, ICOSxe2x88x92, SLAMxe2x88x92, and 4-1BBxe2x88x92. The expression of these markers can be measured by techniques known to those of skill in the art, including, for example, western blot analysis, northern blot analysis, RT-PCR, immunofluorescence assays, and fluorescence activated cell sorter (FACS) analysis.
The term xe2x80x9cT cell activator,xe2x80x9d as used herein, refers to any compound or factor that is a T cell receptor stimulatory factor, that is, induces T cell receptor signalling. Preferably, the compound or factor also induces co-stimulatory pathways. Non-limiting examples of T cell activators include, but are not limited to, anti-CD3, antibodies (preferably monoclonal antibodies) either alone or in conjuntion with anti-CD28 antibodies (preferably monoclonal antibodies), or mitogens such as, for example, phorbol 12-myristate 13-acetate (PMA), phytohemagglutinin (PHA) or concanavalin-A (Con-A).